The zebrafish is a superb genetic system for studying early vertebrate development and organogenesis. It is also an excellent animal model system for studying human diseases. Several large-scale mutagenesis screens have provided thousands of mutant fish with defects in important biological processes. The genetic mapping and cloning of the responsible genes will be particularly useful for the study of biology and disease. In an effort to provide candidate genes and map positions for the zebrafish genome, we have developed two independent radiation hybrid (RH) panels (T51 and LN54). Over the past three years, we have mapped 10,175 markers including ESTs and genes on the zebrafish T51 RH panel map. We have also contributed about 600 same EST markers to the LN54 map, facilitating the integration of the two RH maps. Using informatics, we have developed web sites that enhance our ability to integrate gene orthologies and map positions. The zebrafish field has recently undergone a major transition with the initiation of large-scale genome sequencing by the Sanger Center. We are collaborating with the Sanger Center and, through this grant, are anchoring BAC contigs to the T51 RH map. We will integrate the T51 map with other genome maps to build the framework for assembly of the genome sequence. We will also maintain RH mapping services for the community until an assembled genome is available. Finally, in anticipation of the completed sequence in 2005, we will develop more useful microsatellite markers for mutant mapping. Efficient strategies for mutant mapping and positional cloning will be developed as part of this proposal. A half YAC library will be created to characterize the telomeres of the zebrafish genome, and this will be provided to the Sanger Center for sequencing. These proposed studies should ensure a successful assembly of the genome based on the mapped genes and markers, and will aid investigators to rapidly isolate mutant genes and to study comparative genomics.